Organogenesis in tissue culture is a critical step for efficient transformation of most plants. Media of controlled auxin/cytokinin ratio govern organogenesis in a wide range of species. Cytokinins were first identified as factors that act synergistically with auxin to promote cell division and act antagonistically to auxin to promote shoot and root initiation from callus cultures. Cytokinins have been implicated in many aspects of plant growth and development, including cell division, shoot initiation and growth, leaf senescence, and photomorphogenesis.
Although most molecular mechanisms of cytokinin action are unknown, overexpression of a histidine kinase CKI1 or cyclin D3 confers cytokinin-independent cell growth on cultured cells of Arabidopsis (Kakimoto, 1996; Riou-Khamlichi et al., 1999), indicating that they may play key roles in cytokinin-signaling.
Transgenic techniques have become a powerful tool to address important biological problems in multicellular organisms, and this is particularly true in the plant field. Many approaches that were impossible to implement by traditional genetics can now be realized by transgenic techniques, including the introduction into plants of homologous or heterologous genes, with modified functions and altered expression patterns. The success of such techniques often depends upon the use of markers to identify the transgenic plants and promoters to control the expression of the transgenes.
Selectable markers are widely used in plant transformation. Historically such markers have often been dominant genes encoding either antibiotic or herbicide resistance (Yoder and Goldsbrough, 1994). Although such markers are highly useful, they do have some drawbacks. The antibiotics and herbicides used to select for the transformed cells generally have negative effects on proliferation and differentiation and may retard differentiation of adventitious shoots during the transformation process (Ebinuma et al., 1997). Also, some plant species are insensitive to or tolerant of these selective agents, and therefore, it is difficult to separate the transformed and untransformed cells or tissues (Ebinuma et al., 1997). Further, these genes are constitutively expressed, and there are environmental and health concerns over inserting such constitutively expressed genes into plants which are grown outside of a laboratory setting (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992).
One marker which is neither an antibiotic nor a herbicide is the ipt gene. This gene encodes isopentenyltransferase which is used in cytokinin synthesis (Barry et al., 1984). Overproduction of cytokinins results in the overproduction of shoots (Barry et al., 1984). This overproduction of shoots can result in a phenotype having a large number of shoots (hereafter xe2x80x9cshooty phenotypexe2x80x9d). This phenotype can be used as a marker (Ebinuma et al., 1997). A chimeric ipt gene under the control of the cauliflower mosaic virus (CaMV) promoter has been introduced into cells of potato (Ooms et al., 1983), cucumber (Smigocki and Owens, 1989), and several Nicotiana species (Smigocki and Owens, 1988) and these transgenic cells proliferated and exhibited an extreme shooty phenotype and loss of apical dominance in hormone-free medium. Studies have shown that in plants transformed with ipt to overproduce cytokinins, the cytokinins work only locally as a paracrine hormone (Faiss et al., 1997). One problem with the use of ipt as a marker is that the resulting transgenic plants lose apical dominance and are unable to root due to overproduction of cytokinins (Ebinuma et al., 1997).
Ebinuma et al. (1997) developed one method to use the ipt marker and to overcome the problems noted above. They developed a vector in which the ipt gene was inserted into a plasmid which included the transposable element Ac. The construct included the T-DNA (portion of the Ti plasmid that is transferred to plant cells) and the 35S CaMV promoter. This construct was transformed into A. tumefaciens. Leaf segments were inoculated with the transformed bacteria and grown on nonselective media. Abnormal shoots with an extra shooty phenotype were selected and cultivated further for six months. From these, several normal shoots grew. Some of these were a result of the transposable element Ac having excised from the genome along with the ipt gene. This was determined by DNA analysis. Some of these few plants retained the other necessary markers which had also been included in the plasmid. This method therefore overcomes the problems of having a constitutively expressed ipt gene present. Unfortunately, this method requires many months of cultivation and results in only a few plants which have lost the ipt gene. Ebinuma et al. (1997) report that 6 months after infection the frequency of marker free plants was 0.032%.
The gene CKI1 was more recently identified (Kakimoto, 1996). Overproduction of this gene in plants results in plants which exhibit typical cytokinin responses, including rapid cell division and shoot formation in tissue culture in the absence of exogenous cytokinin (Kakimoto, 1996). The CKI1 gene can be used as a selectable marker in a manner similar to ipt, i.e., the CKI1 gene can be put under the control of a promoter and overexpressed in transgenic plant cells thereby inducing shoot formation in the absence of exogenous plant hormones. Such shoots can be excised thereby obtaining transgenic plants. Such shoots, obtained either from cells transformed with ipt or CKI1, cannot be made to grow normally while the cells are expressing these transgenes. The knotted gene and knotted-like genes are a third group of genes which when overexpressed can lead to ectopic production of adventitious shoots (Chuck et al., 1996; Lincoln et al., 1994). These can be used as selectable markers in the same manner as the ipt and CKI1 genes.
Besides the use of markers to identify transgenic plants, the use of promoters to control the transgenes is a normal part of such experiments. In most experiments, the transgenes are transcribed from a strong promoter, such as the 35S promoter of the cauliflower mosaic virus (CaMV). However, a more flexible gene expression system is needed to extract greater benefits from transgenic technology. Good inducible transcription systems are desired because transgenic plants with inducible phenotypes are as useful as conditional mutants isolated by traditional genetics. In this regard, several induction systems have been reported and successfully used (Ainley and Key, 1990; Gatz et al., 1992; Mett et al., 1993; Weinmann et al., 1994). Among these, the tetracycline-dependent expression systems are the most commonly used (for review, see Gatz, 1996). See Zuo and Chua (2000) for a review of chemical-inducible systems for regulated expression of plant genes.
The glucocorticoid receptor (GR) is a member of the family of vertebrate steroid hormone receptors. GR is not only a receptor molecule but also a transcription factor which, in the presence of a glucocorticoid, activates transcription from promoters containing glucocorticoid response elements (GREs) (for reviews, see Beato, 1989; Picard, 1993). It had been considered that the GR system could be a good induction system in plants because it is simple, and glucocorticoid itself does not cause any pleiotropic effects in plants. Schena et al. (1991) demonstrated that a system comprising GR and GREs could work in a transient expression system with cultured plant cells. It had been reported that the hormone-binding domain (HBD) of GR could regulate the function of plant transcription factors in transgenic plants (Aoyama et al., 1995; Lloyd et al., 1994). Aoyama and Chua (1997) then demonstrated a general and efficient glucocorticoid-inducible system using GR.
Despite the availability of the markers described above and the systems available for controlling the expression of the markers, the need for improved marker genes still exists. Furthermore, a need exists for improving the efficiency of organogenesis or regeneration of plant cells. The present invention addresses both of these needs.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the text and respectively grouped in the appended List of References.
In one aspect, the invention relates to a gene which is useful as a selectable marker in transgenic plants. This gene is herein named Enhancer of Shoot Regeneration (Esr1). The encoded protein, ESR1, contains an AP2 domain which is found in various transcriptional factors of plants.
In a second aspect, the invention is drawn to a protein (ESR1) encoded by Esr1.
A third aspect of the invention is a method of using ESR1 as a selectable marker. Overexpression of ESR1 in plants or plant cells results in plants or cells which show cytokinin-independent growth. This allows the gene to be used as a selective marker by growing cells transformed with the gene in cytokinin-free medium.
In a fourth aspect of the invention, overexpression of ESR1 in plant cells grown in the presence of cytokinins results in extremely high regeneration efficiency. This aspect of the invention uses the gene not as a marker but as a means to increase the regeneration efficiency of plant cells.
The present invention relates to transgenic plants which include the gene Esr1 (enhancer of shoot regeneration 1). This gene is shown as SEQ ID NO:6 and is nearly identical with a gene reported in GenBank (Accession Number AC007357, gene F3F19.1 consisting of bases 2481-3467 of the GenBank sequence which is shown herein as SEQ ID NO:10). The GenBank sequence differs from the Esr1 gene at four bases and the encoded proteins differ at a single amino acid residue. ESR1 is shown as SEQ ID NO:7 and the GenBank encoded protein is shown as SEQ ID NO:11. Overexpression of Esr1 allows cytokinin-independent growth of the plants or cells which are overexpressing the gene thereby allowing the gene to be used as a marker. Furthermore, overexpression of the gene in cells grown in the presence. of cytokinins results in a high regeneration efficiency, thus allowing the gene to be used in a manner which increases plant regeneration.
Expression of Esr1 can be placed under the control of an inducible promoter. An inducible promoter can be one which can be turned on (induced) to select for cells or plants which have become transgenic but which will not be turned on under natural growth conditions. In this manner the selectable marker gene, although present in the transgenic plants, will be completely silent during the normal growth of the plants and should not interfere with the growth of the plants. Such a silent marker gene may also be more environmentally acceptable than, e.g., having an antibiotic resistance gene marker present wherein said resistance gene is expressed during the normal growth of the plant.
To use ESR1 as a silent marker, Esr1 can be placed in a vector with an inducible promoter and plant cells are then transformed with the vector. The plant cells are grown in the presence of an inducer to turn on expression of ESR1 but in media lacking cytokinins. The absence of cytokinins prevents the growth of nontransformed cells, but cells transformed with the vector will grow despite the lack of cytokinins in the medium because of overexpression of the ESR1 gene. Shoots or calli which grow can be selected and tested to ascertain that they do include the vector. Once transformed shoots or calli are selected, they can be grown under normal conditions in the absence of inducer thereby preventing expression of the ESR1 gene within the vector.
The vector which expresses Esr1 under the control of an inducible promoter can, if desired, include a second gene which will be expressed. This second gene can be under the control of an inducible promoter which is different from that controlling the ESR1 gene or it can be under the control of a constitutive promoter. This second gene can then be induced or produced constitutively in the transformed plants which are grown under normal conditions. The second gene can be any gene desired and can produce a protein which results in a more desirable trait than found in the nontransformed plant. Alternatively, plants can be cotransformed with one vector encoding ESR1 and with a second vector encoding the gene of interest. As the two transgenes will segregate in subsequent transgenic generations, transgenic plants carrying only the gene of interest can be obtained this way.
One inducible promoter under which the Esr1 gene can be placed is the glucocorticoid receptor (Aoyama and Chua, 1997). This has been considered to be a good induction system for plants because glucocorticoid itself does not cause any apparent pleiotropic effects in plants. The use of a steroid receptor domain to regulate gene expression in transgenic plants has been previously reported by Lloyd et al. (1994), who showed that trichome development in Arabidopsis could be successfully controlled by a chimeric protein comprising the glucocorticoid receptor domain and the maize transcriptional regulator R. Tight transcriptional regulation with these systems appears to be dependent on the intramolecular structure of the chimeric protein, especially the relative position between the glucocorticoid receptor domain and the domain whose function is to be regulated. Although the glucocorticoid receptor-regulated promoter is a useful one which can be used together with ESR1, Esr1 can be used together with any inducible promoter which is desired. For a recent review of chemical-inducible systems for regulated expression of plant genes see Zuo and Chua (2000).
In addition to its use as a marker, especially as a silent marker, overexpression of Esr1 in the presence of cytokinins results in enhanced regeneration efficiency. This result allows one several options for increasing yields of transformed plants. Vectors can be prepared which contain both a gene of interest as well as Esr1. Plant cells are then transformed with these vectors and the cells are cultured. In a first method, the cells can first be cultured on media lacking cytokinins but including an inducer of the ESR1 gene and the shoots and calli which grow can be selected and then grown normally. In a second method, the cells can be grown on media with cytokinins and with an inducer of Esr1. This second method allows greater regeneration efficiency thereby resulting in more shoot or callus formation. If desired, this second method can be performed with a vector comprising yet a third gene which can encode a selectable marker, e.g., an antibiotic resistance gene. In this method, a vector comprising Esr1 under the control of an inducible promoter, the antibiotic resistance marker gene, and the gene of interest is used to transform cells. The cells are then grown on a medium with an inducer of Esr1, an antibiotic, and cytokinins. The expression of Esr1 results in enhanced regeneration efficiency while the antibiotic resistance gene acts as a selectable marker. The gene of interest will be present in the selected shoots.
The vectors to be used in forming transgenic plants can include a chemically inducible promoter such as the glucocorticoid promoter which will activate the selectable marker but can include any other desired promoter in place of or in addition to the GR system promoter. If desired, any other gene of interest can also be put under control of the inducible promoter such that the gene could be turned on whenever desired. Such a gene need not be a marker.